VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

RGP031The ratio of autoinducers determines Quorum Sensingregulated phenotypes in Vibrio harveyiC. Anetzberger*, N. Stambrau, K. JungDepartment of Biology I, Microbiology, Ludiwg-Maximilians-Unviersity,Munich, GermanyQuorum Sensing (QS) plays an important role in regulating gene expressionin bacterial populations. This intercellular communication through lowmoleculardiffusible molecules, called autoinducers (AIs), enables singlecells to coordinate their behaviour within a population. Vibrio harveyiproduces three AIs which are recognized by three hybrid sensor kinases.Information is transduced via phosphorelay to LuxU and subsequently toLuxO. At low AI concentration the intracellular concentration of phospho-LuxO is high which in turn induces transcription of four regulatory sRNAs.These sRNAs destabilize upon interaction with Hfq the mRNA of LuxR.LuxR induces or represses QS-dependent genes.The extracellular concentration of the three AIs of V. harveyi, HAI-1, CAI-1and AI-2, was monitored in a growing culture over time. According to thedistribution of AIs and QS regulated phenotypes three stages could bedistinguished. In the early exponential growth phase only AI-2 wasdetectable, and bioluminescence was induced (stage 1). In the lateexponential growth phase both HAI-1 and AI-2 reached their maximalvalues, and bioluminescence further increased (stage 2). In the stationarygrowth phase HAI-1 and AI-2 were adjusted to equal concentrations, andCAI-1 was detectable (stage 3). These stages are consistent with in vitrophosphorylation data. The influence of AIs on the in vitro reconstructedsignaling cascade consisting of all three hybrid sensor kinases, LuxP andLuxU was tested. The presence of AI-2 inhibited LuxU phosphorylation by61%, while the additional presence of HAI-1 revealed an inhibition of LuxUphosphorylation by 92%. When all three AIs were present, phosphorylationof LuxU was completely prevented. The data suggest cooperative behaviourof the QS receptors that allows a very sensitive response to various ratios ofexternal AIs.RGP032Analyzing the extent of FtsH-dependent proteolysis bysubstrate trappingK. Westphal*, S. Langklotz, F. NarberhausFaculty of Biology and Biotechnology, Department of Microbial Biology,Ruhr-University, Bochum, GermanyProteolysis is a wide-spread mechanism to ensure the sensitive balance ofregulatory and metabolic proteins at certain conditions. In Escherichia coli,five ATP-dependent proteases are responsible for the specific degradation ofproteins. Among these, FtsH is the only membrane-bound and essentialprotease. Besides the quality control of membrane proteins and SsrA-taggedproteins, the most important role of FtsH is the degradation of regulatoryproteins in the cytosol. For example, FtsH is involved in the heat shockresponse by proteolysis of the heat shock sigma factor RpoH. The essentialfunction of FtsH is the control of LPS biosynthesis by degradation of theLpxC and KdtA enzymes [1, 2].Compared to other proteases, the numbers of identified FtsH-substrates islimited. To find new substrates, a comparative substrate trapping approachwas used. An FtsH-trap version carrying a mutation in the proteolytic center(FtsH_H417Y) was constructed and expressed in E. coli. Substrates arepredicted to be unfolded and translocated into the proteolytic chamber of theFtsH-trap protein. Protease-substrate complexes were co-purified, separatedby 2D PAGE and subjected to mass spectrometry. We identified 12 putativesubstrates of FtsH, among them the known substrate LpxC, validating thismethod as a powerful tool to identify new protease substrates. The list ofputative substrates of FtsH includes proteins with a variety of cellularfunctions. For example, the phage shock protein PspA, the anti sigma factorof RpoD (Rsd), the key enzyme of histidine biosynthesis (HisG) or theuncharacterized and putative protein YfgM co-purified with FtsH_H417Y.First degradation experiments revealed a growth phase-dependentproteolysis of YfgM. Using this experimental setup, we set out to broadenthe understanding of the physiological role of FtsH-dependent proteolysis.[1] Führer, F. et al (2006):The C-terminal end of LpxC is required for degradation by the FtsHprotease. Mol. Microbiol. 59: 1025-1036.[2] Katz, C. and E. Z. Ron (2008): Dual role of FtsH in regulating lipopolysaccharide biosynthesis inEscherichia coli. J Bacteriol. 190: 7117-7122.RGP033Analysis of new P xyl/tet promoters for Tet-ON and Tet-OFF regulation in Staphylococcus aureusS. Mayer* 1 , L. Helle 1 , M. Kull 1 , M.-E. Zelder 1 , R. Bertram 1Institute of Microbiology and Infection Medicine (IMIT), Eberhard-Karls-University, Tübingen, GermanyInducible gene expression systems are useful tools for investigating genefunctionrelationships. The tetracycline-dependent gene expression system(tet system) is based on the regulator TetR which binds to its cognate DNAsequence tetO that is embedded in one or more copies within the promoterregion. P xyl/tet is the most prominent promoter for tet target gene control inStaphylococcus aureus. In Tet-ON architectures, expression of a target geneis repressed by TetR and induced upon addition of an effector such asanhydrotetracycline (ATc) which causes TetR detachment from tetO. Bycontrast, reverse TetR variants only bind to tetO upon interaction with ATc.Tet-OFF systems employing reverse TetR enable rapid silencing of a targetgene by adding this compound.Transcriptional control of the nuclease 1 gene (nuc1) of S. aureus SA113 ina pRMC2 vector system (Corrigan and Foster, 2008) under control of TetRhas shown leakiness under non-induced conditions. Thus a second tetO sitewas inserted, creating the vector pRAB11. Semi quantitative evaluation ofnuclease activity on DNA-containing media indicated enhanced repressioncapabilities of pRAB11. Using lacZ as reporter gene downstream of theP xyl/tet promoter, β-galactosidase measurements verified that the pRAB11vector system enables tighter repression, however at the cost of slightlylower expression levels compared to pRMC2.Since P xyl/tet is a very strong promoter in S. aureus, a promoter pool wasgenerated by randomly mutating up to six conserved positions of the -35 and-10 regions of P xyl/tet to gain different expression levels of target genes in thepRAB11 vector system. 16 different P xyl/tet variants, exhibiting one to threenucleotide exchanges, were characterised in β-galactosidase assays. All ofthem displayed weaker transcriptional potency. Three of them indicatedintermediate expression levels in the induced state accompanied with tighterrepression compared to the wildtype P xyl/tet promoter. This makes pRAB11and its promoter derivatives suitable vector systems for tet regulation instaphylococci, tailored to the specific transcriptional requirements of targetgenes of choice.RGP034The two-component system YehU/YehT of Escherichiacoli - further insights into its transcriptional regulation.S. Behr*, L. Fried, T. Kraxenberger, K. JungCenter for Integrated Protein Science Munich (CiPSM), Biology I -Microbiology, Ludwig-Maximilians-University, Munich, GermanyTwo-component systems (TCS) are the predominant signal transductionsystems in prokaryotes and consist of at least two components: a membraneintegratedhistidine kinase (HK) which senses a stimulus and transduces it ina cellular signal by autophosphorylation, and a response regulator (RR) withDNA-binding activity. Whereas most TCS in Escherichia coli are wellcharacterized, little is known about the YehU/YehT system. The membraneintegratedHK YehU has a GAF-domain, and the highly conserved inputdomain is structurally similar to the input domain of LytS, a potential sensorfor murein subunits in Gram-positive bacteria. YehT posseses a CheY-likereceiver domain and a LytTR DNA-binding domain. The structure of AgrA,a RR with a LytTR DNA-binding domain, represents a novel DNA-bindingtype.When information on the environmental signal is lacking, overproduction ofRRs provides an alternative approach to identify target genes. Thus, the RRsYehT and KdpE, respectively, were overproduced in E. coli, and acomparative transcriptome analysis revealed several target genes.Transcriptional analysis via Northern Blot hybridization using differentstrains and derivatives of YehT and electromobility shift assays confirmedthat only yjiY is under direct transcriptional control of YehU/YehT. TheYehT-binding site was further narrowed by DNase I footprinting.An emerging theme in the field of TCS signaling is the discovery ofauxiliary factors. In vivo protein-protein interaction studies unraveled theauxiliary protein YehS that interacts with YehU and YehT. On the otherhand, bioinformatic tools link YehU/YehT with the YpdA/YpdB twocomponentsystem. Therefore, we hypothesize that the YehU/YehT/YehSsystem is embedded in a signaling network together with the YpdA/YpdBHK/RR system.spektrum | Tagungsband 2011